The inventors first found that ISFETs can be used to monitor local fluctuations in ionic charge corresponding with discrete chemical events, more particularly for example proton release associated with individual nucleotide insertion at the end of an oligonucleotide chain. Monitoring of individual nucleotide insertions by a pH sensitive ISFET may be utilised in DNA sequencing based on conventional Sanger method DNA sequencing and in identifying allelic variants, e.g. single nucleotide polymorphisms (SNPs), relying on detecting extension of oligonucleotide primers designed to target specific nucleic acid sites. The inventors further realised that protons are also a PCR product and that qPCR may therefore also be achieved by ISFET monitoring of proton release, preferably in a low reaction volume chamber
DNA sequencing methods have remained largely unchanged in the last 20 years [Sterky and Lundberg, ‘Sequence analysis of genes and genomes’, J. Biotechnology (2000) 76, 1-31]. The Sanger method is a well-known method of DNA sequencing, and comprises DNA synthesis with termination of DNA replication at points of di-deoxynucleotide insertion. The DNA synthesis is followed by electrophoresis of the synthesised DNA to separate DNA molecules according to their mass to charge ratios, thereby allowing determination of the DNA sequence. A disadvantage of the Sanger method is that electrophoresis is complex, costly and hazardous. It is an object of the present invention to provide a sensing apparatus and method whereby Sanger-type sequencing employing di-deoxynucletide triphosphates can be carried out without need for separation of extended oligonucleotide strands. However, as indicated above, the invention can be applied more broadly to monitoring of any chemical event which will give rise to a fluctuation in ionic charge, e.g. proton release. Sensing devices comprising a pH sensitive ISFET are now also proposed for use in carrying out label free qPCR in small (nano) volumes.